JP2012507998A - Test method to estimate neutralization of asparaginase activity - Google Patents

Abstract

A method for determining in vitro the presence of a factor that neutralizes asparaginase activity in a sample of blood, plasma, serum or derived medium that may contain a neutralizing factor, obtained from said patient, comprising: Mixing a sample with asparaginase, incubating the mixture, and then measuring residual asparaginase activity in the mixture to determine or quantify the presence of a neutralizing factor. A method for estimating the efficacy of treatment with asparaginase.
[Selection] Figure 1

Description

  The present invention relates to a test method for diagnosing a patient's ability to respond to treatment with the therapeutic enzyme asparaginase. The invention particularly relates to a test method for estimating the effectiveness of treatment with asparaginase in a particular patient.

  L-asparaginase is an essential component of the chemotherapy protocol that has been used for over 30 years for the treatment of acute lymphoblastic leukemia. Its mechanism of action is based on the hydrolysis of the plasma amino acid asparagine, an essential element for the neoplastic growth of lymphoblasts. In contrast to normal cells, cancerous lymphoblasts are unable to produce their own asparagine themselves and are dependent on extracellular sources. Treatment with asparaginase depletes this essential component from them and thus kills them.

  Enzymes prepared from microorganisms are currently commercially available in three forms: the first two are derived from the bacterial sources Escherichia coli and Erwinia chrysanthemi, the third is the natural asparaginase of E. coli. It is obtained by covalently binding polyethylene glycol to (PEG-asparaginase).

  Treatment with asparaginase is associated with a number of complications related to the immunogenicity of this enzyme, despite its considerable anti-leukemic efficacy. These complications may be reflected in clinical manifestations or asymptomatic inactivation.

  A number of authors have reported hypersensitivity reactions that vary in severity from moderate allergic reactions to anaphylactic shock (Wang et al., Journal of Immunological Methods 239 (2000) 75-83). When this type of reaction, seen with the three forms of asparaginase, develops, treatment is generally interrupted due to concerns about more severe reactions.

  In addition, asparaginase reveals circulating antibodies with neutralizing properties, which are reflected in increased enzyme clearance by the reticuloendothelial system and reduced therapeutic efficacy (Mueller HJ, Boos J. Crit Rev Oncol / hematol 1998; (28): 97-113). These antibodies have been observed for the three forms of the enzyme (E. coli, Erwinia and PEG-asparaginase), in which case asparaginase therapy objectives, i.e. achieving rapid and complete long-term plasma asparagine depletion, are not realized.

This enzyme inactivation by neutralizing factors, mainly antibodies, present in the patient's serum is not asymptomatic to the clinician with no clinical signs.
Accordingly, the present invention presents a substantial need for clinicians to seek a freely usable rapid and easy-to-use test for estimating the presence of asparaginase neutralizing factors, primarily antibodies, present in patient serum. They confirmed. With this test, it is possible to adjust the dose of the enzyme or to replace the asparaginase used with other forms of asparaginase that are not or less sensitive to these neutralizing factors. I will.

Several possibilities were considered to monitor patient asparaginase activity:
Plasma L-asparagine assay;
An assay for plasma L-asparaginase activity;
Anti-asparaginase antibody assay.

  Plasma asparagine levels are a key biochemical parameter that reflects the targeted therapeutic effect for asparaginase, ie, rapid, complete and sustained asparagine depletion. Accordingly, several methods have been described for its assay. They are largely based on a combination of separating the components of the sample to be assayed by high performance liquid chromatography and detection by fluorescence analysis or quantification by mass spectrometry. These methods are tedious, require trained personnel, and the cost and time required for them are not compatible with routine clinical use.

  More recently, Verma et al. Described a rapid assay for asparagine based on co-immobilization of L-asparaginase and a colored indicator on various supports (nitrocellulose membrane, silicone gel or calcium alginate beads). . When any of these supports is contacted with a patient serum sample, the immobilized L-asparaginase will degrade the asparagine present. The formation of ammonium associated with this hydrolysis reaction will change the color of the phenol red indicator. Although this method seems to meet the need for ease of use and speed, its authors do not provide demonstrable test data and there are still questions about its accuracy.

  Apart from the lack of a proven method that can be used easily and quickly, plasma L-asparagine assays are limited by the following problems. In vivo, the degradation of asparagine by the action of asparaginase is maintained in opposite equilibrium by physiological asparagine production, whereas in vitro the catalytic action of this enzyme on asparagine will persist. As a direct consequence, when serum samples are taken from patients treated with asparaginase, residual amounts of enzyme will bias the measurement of asparagine levels and become “falsely” below physiological levels (Boos et al ., European journal of cancer (1996) 32, 1544-1550). As a result of this interference in the analytical procedure, there is no correlation with physiological asparaginase activity in the subject patient.

  Several methods for assaying plasma L-asparaginase have been described, the most commonly used method is incubating serum containing L-asparaginase in a buffer containing L-asparaginase and then reacting the reaction. After stopping, it is based on measuring the produced ammonium with a Nessler reagent. Orsonneau et al. (J. L. Orsonneau et al. Ann Biol Clin 2004, 62: 568-72) proposed a faster and more accurate automated variant that could monitor patients treated with plasma L-asparaginase. This method is based on the action of glutamate dehydrogenase; it uses the ammonium produced when L-asparagine is hydrolyzed by L-asparaginase to convert α-ketoglutarate to glutamate.

Glutamate dehydrogenase α-ketoglutarate + NH ₄ ⁺ + NADPH → L-glutamate + NADP + H ₂ O
In this reaction, the amount of NADPH oxidized during the course of the reaction is equivalent to the amount of ammonia contained in the sample, and can be determined by measuring the decrease in optical density. Therefore, the kinetics of ammonium appearance can be monitored and the activity of L-asparaginase can be calculated. This method makes it possible to monitor a patient's L-asparaginase activity, but gives no inferred information regarding neutralization of this enzyme by factors present in the serum.

  Wang et al. Developed a standardized ELISA test for quantifying anti-asparaginase IgG in plasma samples from patients (B. Wang et al., Journal of Immunological Methods 239 (2000) 75-83). This test is within the scope of clinical trials to determine the concentration of anti-asparaginase antibodies in the serum of patients with acute lymphoblastic leukemia who have been treated with L-asparaginase and have or have not developed an allergic reaction. Used (MH Woo et al., Leukemia (1998) 12, 1527-1533). The authors were able to show that the intermediate concentration of anti-asparaginase antibody was higher in patients who developed an allergic reaction, regardless of whether the measurement was performed before or after the allergic reaction occurred. They conclude that the use of such a test to estimate future onset of allergic reactions is beneficial in clinical practice.

Nevertheless, the estimates of such tests are questionable; the range of variation of anti-asparaginase antibody concentrations measured before the development of allergic reactions overlaps, each of which is as follows:
• OD from 0.001 to 0.375 units for patients who later developed an allergic reaction;
• OD from 0.004 to 0.064 units for patients who did not develop an allergic reaction later.

Furthermore, the measurement of antibody concentration gives no information regarding the pharmacological activity of asparaginase in the patient and its possible neutralization by factors present in the serum.
EH Panosyan et al., J. Pediatr. Hematol. Oncol. 2004, 26, 4: 217-226 examined anti-asparaginase antibodies and asparaginase enzyme activity in patient sera. The authors describe an ex vivo neutralization assay performed using patient serum specimens as a source of anti-asparaginase antibodies. Serum specimens were incubated with natural or PEG-asparaginase antigen solutions and residual asparaginase enzyme activity was measured. The authors finally recommended standard monitoring of anti-asparaginase antibodies in clinical settings.

Wang et al., Journal of Immunological Methods 239 (2000) 75-83 Mueller H.J., Boos J. Crit Rev Oncol / hematol 1998; (28): 97-113 Boos et al., European journal of cancer (1996) 32, 1544-1550 J. L. Orsonneau et al. Ann Biol Clin 2004, 62: 568-72 M. H. Woo et al., Leukemia (1998) 12, 1527-1533 E.H. Panosyan et al., J. Pediatr. Hematol. Oncol. 2004, 26, 4: 217-226

  The object of the present invention is therefore to overcome the drawbacks of the prior art and to propose a new method which makes it possible to know at a particular point in time whether a patient has a factor that may neutralize asparaginase activity. is there. This method must have the advantage of being completely putative; that is, the method should not require administering the enzyme to the patient to be diagnosed. It must reflect the patient's ability to respond to any form of asparaginase. Thus, the patient may be a patient who is scheduled to be treated with this enzyme for the first time, or a patient who has been or is currently being treated with asparaginase. Can be tested for the presence of factors that may neutralize the activity of the enzyme used in past or current treatment, thereby knowing and possibly adjusting whether treatment with this enzyme can be resumed or continued It becomes possible. It is also possible to detect the presence of factors that may neutralize the activity of the enzyme under consideration for the patient's treatment, thereby allowing confirmation or exclusion of the use of a particular enzyme.

  This finding will provide the specialist with much more appropriate guidance than the prior art methods on the form of treatment (dose, dosage regimen) or choice of enzyme or its dosage form. The neutralizing factor is not present, or present at a sufficiently low level that it can be treated with the test enzyme, optionally enhancing the dosage or dosing schedule. Alternatively, neutralizing factors are present at a level that is too high for the continuation or initiation of such treatment, in which case the invention is in a form that is less or less sensitive to other forms of enzymes or neutralizing factors. Allowing the testing and / or recommendation of alternative solutions using, for example, enzymes contained in biovectors.

Accordingly, it is an object of the present invention to propose an in vitro method for determining the presence of a factor that neutralizes asparaginase activity in a blood sample from a patient.
The present invention determines the ability of a patient to respond in vitro (i) respond positively to treatment with asparaginase, (ii) not respond to it, or (iii) respond only incompletely. It is also aimed at advocating a method.

  The present invention is a method for estimating the effectiveness of treatment with asparaginase or for estimating the fact that this enzyme will not immediately be subject to substantial inactivation of its activity by neutralizing factors. The purpose is to advocate.

  Thus, the present invention is a method for estimating whether asparaginase may be active in a patient, which may contain a neutralizing factor, obtained from the patient for the presence of a factor that neutralizes asparaginase activity. It relates to a method for determining in vitro in a sample of blood, plasma, serum or derived medium.

  The present invention is a method for determining in vitro the presence of a factor that neutralizes asparaginase activity in a sample of blood, plasma, serum or derived medium that may contain a neutralizing factor, obtained from a patient. A method comprising: mixing a sample with asparaginase; incubating the mixture; and then measuring the residual activity of the asparaginase in the mixture; this comprises a neutralizing factor in the sample and thus in a patient in vivo Reflects the presence of and enables determination or quantification of it. This method allows qualitative and quantitative diagnosis of the presence of asparaginase neutralizing factor in a patient. By neutralizing factor is meant not only an anti-asparaginase antibody but also any other factor capable of inhibiting asparaginase or its enzymatic activity, such as a protease, eg human asparaginyl endopeptidase.

  The invention further relates to a method for estimating whether a particular asparaginase may be active in a particular patient. This method includes a method for determining in vitro the patient's ability to respond to treatment with an asparaginase, wherein the blood, plasma, serum or possibly obtained from the patient may contain factors that neutralize asparaginase activity. A sample of the induced medium is subjected to a method comprising mixing with the asparaginase, incubating the mixture, and then measuring the residual enzyme activity of the asparaginase in the mixture; Reflects the presence of neutralizing factors that may neutralize asparaginase activity in patients with the ability to determine or quantify it and thus determine the patient's ability to respond to treatment with this enzyme. The present invention thus provides a method for testing the effectiveness of asparaginase for a particular patient.

  Depending on the presence or absence of neutralizing factors and, optionally, their levels, the processes and methods of the present invention allow a patient to (i) respond positively to treatment with asparaginase, or (ii) against it Allows determining the likelihood of not responding or (iii) responding incompletely.

The sample may be from a patient currently being treated with asparaginase or a patient who has been treated with asparaginase.
The sample may be from asparaginase or a patient who has not been treated with this asparaginase.

The asparaginase used for the test may or may have been used for treatment performed on the patient.
It may be an enzyme from a different source than the one that one wishes to test in the patient to estimate its effectiveness. Various enzymes can be tested simultaneously or sequentially to determine the optimal treatment for the patient.

  Thus, the present invention provides a method for estimating the effectiveness of treatment with asparaginase or for estimating the fact that this enzyme will not be subject to substantial inactivation by neutralizing factors immediately. To do.

  The invention applies to all forms of asparaginase, for example L-asparaginase. Without limitation, natural enzymes obtained from any bacterial source, such as L-asparaginase produced by E. coli, enzymes produced by Erwinia, mutant enzymes, or modified enzymes, such as PEGylated An enzyme (PEG-asparaginase) can also be mentioned. The enzyme may be of natural, synthetic or recombinant origin. It may be free or contained in a biovector, such as red blood cells.

  The residual activity of asparaginase in the mixture can be measured by adding to the mixture asparagine, preferably L-asparagine, and a reagent system that can detect enzymatic degradation of asparagine by active asparaginase.

According to an advantageous embodiment, the method according to the invention comprises the following steps:
(A) incubating the sample with a known amount of asparaginase;
(B) incubating the mixture with a known amount, preferably an amount of asparagine that produces saturation relative to asparaginase;
(C) incubating the mixture with a reagent system capable of assaying residual enzyme activity;
(D) Qualitatively or quantitatively assess the loss or retention of enzyme activity and determine the correlation between this and the presence or content of a factor that neutralizes asparaginase activity in the sample.

The incubation in step (a) particularly requires 1 to 60 minutes. The enzyme content is in particular from 0.1 to 5 IU / ml.
It may be useful and advantageous to inactivate or remove any trace amounts of active enzyme in the test sample. Thus, according to one feature, prior to step (a), a step (a ₀ ) of removing or inactivating any asparaginase present in the sample is performed.

Alternatively, prior to step (a), a step (a ₀ ) of measuring the baseline content of asparaginase in the sample can be performed, demonstrating that its activity is zero or negligible. Alternatively, its activity can be subtracted from that measured by this method.

As a variant, knowing the half-life of the enzyme administered to the patient prior to the test can wait for the time required between the last dose and the blood sample collection.
According to a particular aspect, following step (a) Antibodies - carrying out step (a ₁₎ to remove the asparaginase immune complexes. This removal can be easily performed by centrifugation, so the mixture involved in step (b) is the supernatant. Preferably, the centrifugation is carried out at 3000-25000 g for 1-30 minutes at the specified speed.

According to one feature, the reagent system is sensitive to the appearance of ammonium ions resulting from the enzymatic degradation of asparagine by asparaginase. Therefore, the presence of the neutralizing factor can be determined or measured by carrying out a reaction that quantitatively consumes ammonium ions. This consumption of ammonium ions can then be quantified, preferably by measuring the decrease in the optical density (absorbance) of the mixture. In particular, the following reaction is carried out:
(1) Asparaginase + asparagine → aspartic acid + NH ₄ ⁺
(2) α-ketoglutarate + NH ₄ ⁺ + NADPH + glutamate dehydrogenase (catalyst) → glutamate + NADP ⁺ + H ₂ O
Incubation with asparagine is preferably carried out with a saturating amount of asparagine (relative to the amount of enzyme introduced), in particular 10-50 mg / ml, for 2-60 minutes.

Incubation with the reagent system preferably takes 3 to 20 minutes.
Accordingly, the present invention provides a method for determining the likelihood that a patient will (i) respond positively to treatment with asparaginase, (ii) not respond to it, or (iii) respond only incompletely. provide. Thus, this can confirm or eliminate the possibility of adopting treatment with the test enzyme, or alter the dosing regimen or decide to treat the patient with a different asparaginase, particularly in a form encapsulated in a biovector. Can be done.

The present invention therefore also relates to a method of treating an asparaginase sensitive condition in a patient, including:
(A) A method for determining in vitro the ability of a patient to respond to treatment with a particular form of asparaginase (especially in a free or modified form), comprising an asparaginase neutralizing factor obtained from the patient A sample of blood, plasma, serum or derived medium that may be mixed with the form of asparaginase, incubating the mixture, and then measuring the residual activity of asparaginase in the mixture and neutralizing Determine activity and (i) the patient's ability to respond positively to treatment with this form of asparaginase, or (ii) not respond to it, or (iii) respond only incompletely And (B) in case (i) the condition is treated with this asparaginase, or in other cases Are treated with other forms of asparaginase.

  According to a preferred embodiment, another form of asparaginase is asparaginase encapsulated or housed in a biovector, preferably encapsulated in erythrocytes. In particular, they are, for example, French patent application no. Red blood cells prepared by cell lysis and resealing according to the teachings of 0408667.

  Conditions in which this method may be beneficial include in particular leukemias such as acute lymphoblastic leukemia. Solid tumors (WO 2007/103290), particularly pancreatic cancer and ovarian cancer, may also be mentioned, but are not limited to these.

FIG. 1 is a graph showing the results of measuring seven constant L-asparaginase concentrations (range from 0 to 800 IU / L) in three human sera and buffer controls. FIG. 2 is a graph showing the distribution of activity measured for 52 human sera mixed with L-asparaginase at a final concentration of 500 IU / L. FIG. 3 is a graph showing L-asparaginase activity inhibition vs. anti-asparaginase IgG concentration. FIG. 4 is a graph showing L-asparaginase activity inhibition vs. low anti-asparaginase IgG concentration. FIG. 5 is a graph showing L-asparaginase activity inhibition vs. L-asparaginase concentration. FIG. 6 is a graph showing the test results of 57 human sera from 17 patients treated with L-asparaginase.

The invention will now be described in more detail by way of examples; these are illustrative of the invention and are not limiting.
Example 1: Immunization of rabbits with L-asparaginase To obtain pre-immune serum, several milliliters of serum are collected from the rabbit before the first immunization. The rabbits are then injected 4 times with 500 μg L-asparaginase (Kidrolase®, OPI-EUSA Limonest France). Serum is collected between the first and second immunizations and between the second and third immunizations. Finally, whole serum is collected after final immunization and stored at -20 ° C. The final serum is characterized by its total protein concentration (Biulet method) and its total immunoglobulin concentration.

Example 2: Purification of Rabbit Total IgG Purification of rabbit total IgG from final serum containing anti-asparaginase IgG is performed with Kit pure 1A (# PURE1A) from Sigma. In summary, prior to IgG purification, 2 ml of serum is clarified by centrifugation or filtration through a 0.45 μm filter. 4 ml of “binding buffer” is then added to 2 ml of clarified serum. The mixture is passed through the column and then eluted according to the protocol recommended by Sigma. To exchange the elution buffer with PBS so that they can be injected into animals, total IgG is centrifuged in a threshold column of 10,000 daltons.

Example 3a: Measurement of inhibition of intermediate serum on L-asparaginase enzyme activity (mixture assay)
The assay for L-asparaginase was performed according to the protocol published below: Orsonneau et al., “Automatic kinetic assay of plasma L-asparaginase activity in therapeutic monitoring of acute lymphoblastic leukaemias”, Ann Biol Clin, 62: 568-572.

  First, intermediate serum (collected between the first and second immunizations) was used to discuss the measurement of inhibition of enzyme activity. L-asparaginase at a concentration of 2 IU / ml is used. The enzyme is preincubated with several serum dilutions at 37 ° C. for 15 minutes and then the enzyme activity is measured in the mixture. The results are shown in Table 1.

  Table 1 summarizes the measured values of the residual enzyme activity of L-asparaginase in the mixture (test tubes 2 to 7). Rabbit serum inhibits the enzyme activity of L-asparaginase: the greater the serum dilution, the weaker the inhibition.

Tube 1 constitutes a control and shows that incubation of enzyme alone for 15 minutes at 37 ° C. does not affect its enzyme activity.
Example 3b: Measurement of inhibition of intermediate serum on L-asparaginase enzyme activity (supernatant assay)
Intermediate serum (collected between the first and second immunizations) was used.

  To mimic the phagocytosis of antigen-antibody complexes by the reticuloendothelial system, the L-asparaginase / serum mixture is incubated at 37 ° C. for 15 minutes and then centrifuged at 17500 g for 10 minutes at 4 ° C. to remove immune complexes. To do. The enzyme activity is assayed in the supernatant. The results are shown in Table 2.

  To examine the specificity of the interaction between L-asparaginase and the anti-asparaginase antibody present in the serum, a control was added in which the serum was replaced with preimmune serum. This demonstrates that over 90% of the enzyme is not involved in interaction with non-specific antibodies.

For dilutions 1/4, 1/16, 1/32 and 1/128, the antibody present in the serum interacted with all enzymes.
Example 4: Determination of inhibition of rabbit total IgG from L-asparaginase enzyme activity The same experiment as shown in Example 3b was performed using rabbit total IgGs and L-asparaginase at a concentration of 1.25 IU / ml. . The results are shown in Table 3.

  Rabbit total IgG containing anti-asparaginase IgG initiates interaction with L-asparaginase from a dilution of 1/64 and completely inhibits enzyme activity (99.02% of the putative enzyme).

Example 5: Inactivation of free L-asparaginase injected into mice with rabbit total IgG Experiments were set up to investigate inhibition of L-asparaginase by anti-asparaginase IgG in vivo on mice (16 mice).

The experimental conditions were as follows:
-Dose 100 IU / kg L-asparaginase, corresponding to a circulation of 1.25 IU / ml in 25 g mice-Injection of 7.5 [mu] g rabbit total IgG.

  An injection of IgG or PBS is performed 20 minutes prior to the injection of free L-asparaginase or L-asparaginase (Asp-RBC) encapsulated in mouse erythrocytes. The mice are then sacrificed 6 hours after this final injection and blood is collected.

  The L-asparaginase activity is then assayed in plasma and RBC. The numerical values obtained are summarized in Table 4.

  Assay of L-asparaginase in RBCs of mice dosed with Asp-RBC detects 0.798 and 0.879 IU / ml in the presence of IgG or PBS, respectively. Therefore, IgG present in plasma did not have an inhibitory effect on the encapsulated enzyme. In the plasma of these same mice, free L-asparaginase injected with Asp-RBC (in fact, as little as 10% of the free enzyme is still outside RBC) is inhibited in the presence of IgG ( 0.013 IU / ml) and retains activity in the presence of PBS (0.126 IU / ml).

  When free L-asparaginase was injected, its activity was inhibited by IgG (0.002 IU / ml), whereas PBS injection had no effect on enzyme activity (0.417 IU / ml). . Considering the half-life of free L-asparaginase (10 hours), the measured value of L-asparaginase 0.417 IU / ml plasma corresponds to the free enzyme residual activity 6 hours after the injection of free enzyme.

  The plasma concentration of L-asparagine was measured in the plasma of 14 test mice (2 mice could not be employed because the volume was too small). The results are shown in Table 5.

L-asparagine depletion was complete when mice were treated with Asp-RBC or when mice were administered free L-asparaginase in the presence of PBS.
Only mice treated with free L-asparaginase in the presence of IgG have a plasma L-asparagine concentration of 28.09 μM. Therefore, this enzyme was inhibited by IgG in plasma.

Example 6: Measurement of Rabbit Total IgG Inhibition on Enzyme Activity of PEG-Asparaginase Rabbit total IgG containing anti-asparaginase IgG was tested against PEG-asparaginase (Sigma # A5336). The same experiment as shown in Example 4 was performed on PEG-asparaginase. The results are shown in Table 6.

Rabbit IgG containing anti-asparaginase IgG interacted with PEG-asparaginase. The activity detected in the supernatant is less than 4% of the activity initially mixed with IgG.
Example 7: Testing protocol This protocol is applied to patients under consideration for treatment with a specific asparaginase. A small blood sample is taken from this patient and processed according to routine to obtain a serum sample.

Then follow the procedure below:
(A ₀ ) optionally inactivating or removing any asparaginase present in the serum sample, or measuring residual enzyme activity;
(A) Incubate the sample with 0.1-5 IU / ml asparaginase for 1-60 minutes;
(A ₁ ) Optionally, immune complexes are removed by centrifugation at a specified speed, preferably at 3000-25000 g for 1-30 minutes;
(B) Incubating the mixture from (a) or the supernatant from (a ₁ ) with a saturating amount, in particular 10-50 mg / ml asparagine, for 2-60 minutes;
(C) incubating said mixture with a reagent system (α-ketoglutarate, NADPH, glutamate dehydrogenase) capable of detecting residual enzyme activity, in particular for 3-20 minutes;
(D) Qualitatively or quantitatively assess the loss or retention of enzyme activity and determine the correlation between this and the presence or content of asparaginase neutralizing factor in the sample.

Depending on whether step (a ₁ ) is included or not, the level of residual enzyme activity or a threshold of levels can be measured.
If this method shows significant presence of neutralizing factors in already treated patients, alternative therapies using different forms of asparaginase are recommended and applied. In particular, an enzyme encapsulated in erythrocytes is recommended when free or modified forms may be inhibited.

The same protocol can be easily applied to test the potential effectiveness of asparaginase in patients.
Example 8: Effect of patient serum matrix on L-asparaginase activity measurement Three human sera naive to L-asparaginase treatment to ensure that human serum does not interfere with L-asparaginase activity measurement Was mixed with 7 concentrations of L-asparaginase (from 0 to 800 IU / L). Samples were incubated at 37 ° C. for 15 minutes and centrifuged at 17500 g, 4 ° C. for 10 minutes. L-asparaginase activity is examined in the supernatant. As a control, buffer 1 × PBS 4% BSA was mixed with the same concentration of L-asparaginase. The results are shown in Table 7.

^* : Not measured.
There was no human serum interference with the L-asparaginase activity measurement (contrast with buffer control; see Table 7 and FIG. 1).

Example 9: Effect of patient serum matrix on measurement of L-asparaginase activity and measurement of basal activity of human serum To ensure that human serum does not interfere with the measurement of L-asparaginase activity, L-asparaginase treatment was performed. In contrast, 52 naive human sera were mixed with L-asparaginase at a final concentration of 500 IU / L. Samples were incubated at 37 ° C. for 15 minutes and centrifuged at 17500 g, 4 ° C. for 10 minutes. L-asparaginase activity is examined in the supernatant. As a control, buffer 1 × PBS 4% BSA was mixed with the same final concentration of L-asparaginase. The results are shown in Table 8.

  Table 8: Measurement of l-asparaginase activity for 52 human sera supplemented with L-asparaginase at a final concentration of 500 (IU / L)

  The average activity measured for 52 human sera is 473 IU / L with a standard deviation (SD) of 13.4 IU / L. This average activity measured on serum is not significantly different from control activity. All numerical values are within tolerance: only 3 out of 52 measurements are outside the confidence range [average-2SD; average + 2SD]. The distribution of activity measured according to the assayed serum indicates that L-asparaginase activity is not affected by the matrix serum (FIG. 2).

  To test for the absence of an enzyme activity signal in human serum, 25 human sera naive to L-asparaginase treatment were assayed for l-asparaginase activity.

  Table 9: Measurement of l-asparaginase activity on 25 human sera naive to L-asparaginase treatment

  For each of the 25 human sera assayed, the L-asparaginase activity is close to zero. The measured average activity is 0.88 IU / L with a standard deviation of 1.27 IU / L. Since the maximum activity measured is 5 IU / L for serum 17, which is 1% of the activity measured for serum mixed with a final concentration of 500 IU / L L-asparaginase, this basal activity signal is L-asparaginase Does not appear to affect activity measurement.

Example 10: Inhibition of L-asparaginase activity by human serum spiked with anti-asparaginase IgG Two human sera naive to L-asparaginase treatment were pooled and the concentration range 1-100 μg / mL (1, 2, 5, 10 , 20, 40, 80, 100 μg / mL) was spiked with anti-asparaginase IgG. Anti-asparaginase IgG was obtained as described in Examples 1 and 2.

  500 IU / L L-asparaginase was then added. Samples were incubated at 37 ° C. for 15 minutes and centrifuged at 17500 g, 4 ° C. for 10 minutes. Residual L-asparaginase activity was measured in the supernatant. As a control, buffer 1 × PBS 4% BSA was used instead of human serum pool. The results are shown in Table 10 and FIG.

^* : Not measured.
When L-asparaginase is mixed with increasing concentrations of anti-asparaginase IgG (specific IgG), complete enzyme activity inhibition occurs in human serum or buffer control at concentrations of 20 μg / mL or higher. When the enzyme is mixed with 5-20 μg / mL anti-asparaginase IgG, partial inhibition occurs. Less than 5 μg / mL anti-asparaginase IgG does not inhibit L-asparaginase activity.

No inhibition is seen when L-asparaginase is incubated with non-specific IgG (see control IgG added in Table 10).
To improve the inhibitory response of 10-20 μg / mL concentration of anti-asparaginase IgG to L-asparaginase activity, experiments were performed with IgG ranging from 8 to 22 μg / mL. Add L-asparaginase as usual to a final concentration of 500 IU / L. All samples were incubated at 37 ° C. for 15 minutes and centrifuged at 17500 g, 4 ° C. for 10 minutes. Residual L-asparaginase activity is measured in the supernatant. This assay is performed on a human serum pool. The results are shown in Table 11 and FIG.

Complete inhibition of L-asparaginase activity is seen at an IgG concentration of 16 μg / mL. Below 16 μg / mL, L-asparaginase inhibition is partial.
The opposite reaction was examined: a constant concentration of anti-asparaginase IgG (13,64 μg / mL, corresponding to 80% inhibition) was mixed with L-asparaginase in a concentration range of 500 to 10000 lU / L. Samples were incubated at 37 ° C. for 15 minutes and centrifuged at 17500 g, 4 ° C. for 10 minutes. Residual L-asparaginase activity is measured in the supernatant. The results are shown in Table 12 and FIG.

  The higher the L-asparaginase concentration, the less the constant concentration of IgG (13.64 μg / mL) inhibits its activity. A certain amount of anti-asparaginase IgG inhibits a certain amount of L-asparaginase. Thus, inhibition of L-asparaginase activity is dose dependent.

Example 11: Examination of 57 human sera from 17 patients treated with L-asparaginase To confirm that this assay is capable of quantifying neutralization of asparaginase in patients, in treatment with L-asparaginase 57 human sera sampled from 17 patients with acute lymphoblastic leukemia were tested according to the test protocol described in Example 7. Samples were taken at various points in the course of treatment and measurements of residual L-asparaginase enzyme activity were performed to demonstrate that this activity was negligible and would not interfere with the testing procedure.

  The serum was then mixed with a final concentration of 500 IU / 1 L-asparaginase and incubated at room temperature for 15 minutes. The L-asparaginase activity was then measured before and after centrifugation at 7800 rpm for 6 minutes. As a control, buffer 1 × PBS 4% BSA was mixed with a final concentration of 500 IU / 1 L-asparaginase. The results are shown in Table 13 below and FIG.

  Table 13: Examination of 57 human sera from 17 patients being treated with L-asparaginase

ND: not measured;
NA: Not applicable.
All samples have negligible residual asparaginase activity, the highest residual activity being 15 IU / L, which is only 3% of the theoretical loading of L-asparaginase and should not interfere with the test protocol. The asparaginase activity measured for the control is higher than expected (655, 636 and 661 IU / L for the three controls, respectively, compared to the expected 500 IU / L). The percent inhibition of asparaginase activity was calculated based on the enzyme activity measured for the control. The fact that many numbers of percent inhibition are negative points out bias in the measurement operation.

  The percent inhibition of asparaginase activity is higher after centrifugation, suggesting that some immune complexes formed between asparaginase and anti-asparaginase antibody were removed by the centrifugation step.

  In the 17 patients assayed, 14 show inhibition of asparaginase activity by factors present in their serum (FIG. 6). High patients have a percent inhibition greater than 20%, but only three patients have a percent inhibition greater than 40% (FIG. 6).

Claims (17)

  A method for estimating whether asparaginase may be active in a patient, the presence of a factor neutralizing asparaginase activity obtained from said patient, blood, plasma, serum possibly containing neutralizing factor Or a method for determining in vitro in a sample of induced medium.   The method of claim 1, comprising mixing the sample with asparaginase, incubating the mixture, and then measuring the residual activity of the asparaginase in the mixture to determine or quantify the presence of a neutralizing factor.   Method according to claim 1 or 2, characterized in that the sample is from a patient currently treated with asparaginase or from a patient treated with asparaginase.   4. The method according to any one of claims 1 to 3, wherein the measurement of residual asparaginase activity in the mixture is carried out by adding asparagine and a reagent system suitable for assaying residual enzyme activity to the mixture. The method of claim 4 comprising the following steps:
(A) incubating the sample with a known amount of asparaginase;
(B) incubating the mixture with a known amount of asparagine;
(C) incubating the mixture with a reagent system suitable for assaying residual enzyme activity;
(D) Qualitatively or quantitatively assess the loss or retention of enzyme activity and determine the correlation between this and the presence or content of asparaginase neutralizing factor in the sample. Before stage (a), comprising (a ₀₎ to remove or inactivate any of asparaginase may be present in the sample The method of claim 5. The method according to claim 5, comprising the step (a ₀ ) of measuring the baseline content of asparaginase in the sample before step (a).   8. A method according to any one of claims 4 to 7, wherein the reagent system is sensitive to the appearance of ammonium ions resulting from the enzymatic degradation of asparagine by asparaginase.   The method of Claim 8 using the reaction which consumes ammonium ion quantitatively.   10. A method according to claim 9, wherein the quantitative consumption of ammonium ions is measured by measuring the decrease in optical density of the mixture. The method according to any one of claims 4 to 10, wherein the following reaction is used:
(1) Asparaginase + asparagine → aspartic acid + NH ₄ ⁺
(2) α-ketoglutarate + NH ₄ ⁺ + NADPH + glutamate dehydrogenase (catalyst) → glutamate + NADP ⁺ + H ₂ O   Determining the patient's ability to respond (i) respond positively to treatment with this form of asparaginase, or (ii) not respond to it, or (iii) respond only incompletely. A method according to any one of the preceding claims comprising.   13. The method of claim 12, comprising a decision to treat the patient with any other asparaginase for cases (ii) and (iii).   13. The method of claim 12, comprising for case (i) the decision to treat the patient with this asparaginase.   14. The method according to claim 13, wherein the other asparaginase is an asparaginase housed in a biovector, preferably encapsulated in erythrocytes. A method of treating a patient's asparaginase sensitive condition, including:
(A) A method for determining in vitro the ability of a patient to respond to treatment with a particular form of asparaginase (especially in a free or modified form), comprising an asparaginase neutralizing factor obtained from the patient To a sample of blood, plasma, serum or derived medium that may be mixed with the form of asparaginase, incubating the mixture, and then the residual activity of asparaginase in the mixture, and (i) A method comprising determining a patient's ability to respond positively to treatment with a form of asparaginase, or (ii) not responding to it, or (iii) responding incompletely. And (B) in case (i) the condition is treated with this asparaginase or in cases (ii) and (iii) Treatment with other forms of asparaginase.   17. A method according to claim 16, wherein the other form consists of a biovector containing asparaginase, preferably erythrocytes encapsulating asparaginase.